Essentials of Anatomy & Physiology, 4th EditionMartini / Bartholomew

PowerPoint® Lecture Outlines prepared by Alan Magid, Duke University

The Muscular

System

The Muscular

System77BLOTTING TECHNIQUES

Tapeshwar Yadav(Lecturer)BMLT, DNHE,

M.Sc. Medical Biochemistry

Introduction• Concept: reannealing nucleic acids to identify sequence of

interest.

• Separates DNA/RNA in an agarose gel, then detects specific bands using probe and hybridization.

• Hybridization takes advantage of the ability of a single stranded DNA or RNA molecule to find its complement, even in the presence of large amounts of unrelated DNA.

• Allows detection of specific bands (DNA fragments or RNA molecules) that have complementary sequence to the probe.

• Size bands and quantify abundance of molecule.

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What is blotting?

Blots are techniques for transferring DNA , RNA

and proteins onto a carrier so they can be

separated, and often follows the use of a gel

electrophoresis. The Southern blot is used for

transferring DNA, the Northern blot for RNA and

the western blot for PROTEIN.

TYPES OF BLOTTING TECHNIQUES

SOUTHERN BLOTTING• Professor Sir Edwin Southern,

Professor of Biochemistry and Fellow of Trinity developed this method in 1975.

• Southern won the Lasker Award for Clinical Medical Research prize for the method of finding specific DNA sequences he developed this procedure at Edinburgh University more than 30 years ago. The technique is known as DNA transfer or 'Southern blotting'

Professor Sir Edwin Southern

Cont….

• This method Involves separation, transfer and hybridization.

• It is a method routinely used in molecular biology for

detection of a specific DNA sequence in DNA samples. The

DNA detected can be a single gene, or it can be part of a

larger piece of DNA such as a viral genome.

Cont….

• Southern blotting combines agarose gel electrophoresis for

size separation of DNA with methods to transfer the size

separated DNA to a filter membrane for probe

hybridization.

• The key to this method is Hybridization.

• Hybridization - Process of forming a double-stranded DNA

molecule between a single-stranded DNA probe and a

single-stranded target patient DNA.

PRINCIPLE

1. The mixture of molecules is separated.

2. The molecules are immobilized on a matrix.

3. The probe is added to the matrix to bind to the

molecules.

4. Any unbound probes are then removed.

5. The place where the probe is connected corresponds to

the location of the immobilized target molecule.

APPARATUS

Whatman 3MM paper nitrocellulosemembrane

Capillary blotting apparatus

Southern Steps1. DNA to be analyzed is digested to completion with a

restriction endonuclease.

2. Electrophoresis to maximally separate restriction fragments in the expected size range. A set of standards of known size is run in one lane of the gel.

3. Blot fragments onto a nitrocellulose membrane.

4. Hybridize with the 32P probe.

5. Autoradiography.

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Steps in southern blotting

1. Digest the DNA with an

appropriate restriction

enzyme.

2.The complex mixture of

fragments is subjected to gel

electrophoresis to separate

the fragments according to

size.

Cont….3.The restriction fragments present

in the gel are denatured with

alkali and transferred onto

4. a nitrocellulose filter or nylon

membrane by blotting.

• This procedure preserves the

distribution of the fragments in

the gel, creating a replica of the

gel on the filter.

Cont….

5.The filter is incubated under

hybridization conditions

with a specific radiolabeled

DNA probe.

• The probe hybridizes to the

complementary DNA

restriction fragment. 

Cont….

6.Excess probe is washed away and the

probe bound to the filter is detected by

autoradiography, which reveals the

DNA fragment to which the probe

hybridized.

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General Scheme for Southern Blot

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APPLICATIONS

• Southern blots are used in gene discovery , mapping,

evolution and development studies, diagnostics and

forensics (It is used for DNA fingerprinting, preparation of RFLP maps)

• identification of the transferred genes in transgenic individuals, etc.

• It is an invaluable method in gene analysis.

• Important for the conformation of DNA cloning results.

• Highly useful for the determination of restriction fragment

length polymorphism (RFLP) associated with pathological

conditions.

APPLICATIONS

• Southern blots allow investigators to determine the

molecular weight of a restriction fragment and to measure

relative amounts in different samples.

• Southern blot is used to detect the presence of a particular

bit of DNA in a sample

• analyze the genetic patterns which appear in a person's

DNA.

• Forensically applied to detect minute quantities of DNA (to

identify parenthood, thieves, rapists etc.)

Southern Application: Diagnosis & detection of genetic diseases.

• Used to diagnose sickle cell-anemia.

• AT base change in the subunit of Hb

Glu Val.

• Development of a 19 residue oligonucleotide probe complementary to sickle-cell gene’s mutated segment.

• Probe hybridizes to DNA from homozygotes of sickle-cell anemia but not from normal individuals.

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‘’This life is a hard fact; work your way through it boldly, though it may be adamantine; no matter, the soul is stronger’’

Northern Blotting

Northern blotting is a technique for detection of specific

RNA sequences. Northern blotting was developed by

James Alwine and George Stark at Stanford University

(1979) and was named such by analogy to Southern

blotting

Steps involved in Northern blotting

1. RNA is isolated from

several biological samples

(e.g. various tissues,

various developmental

stages of same tissue etc.)

* RNA is more susceptible

to degradation than DNA.

Cont……

2. Sample’s are loaded on gel  and

the RNA samples are separated

according to their size on an

agarose gel .

• The resulting gel following

after the electrophoresis run. 

Cont……

3. The gel is then blotted on

a nylon membrane or a

nitrocellulose filter paper

by creating the sandwich

arrangement.

Cont……

4. The membrane is placed in a dish

containing hybridization buffer

with a labeled probe.

• Thus, it will hybridize to the RNA

on the blot that corresponds to

the sequence of interest. 

5. The membrane is washed to

remove unbound probe.

Cont……

6. The labeled probe is detected via

autoradiography or via a

chemiluminescence reaction (if a

chemically labeled probe is used).  In

both cases this results in the formation

of a dark band on an X-ray film.

• Now the expression patterns of the

sequence of interest in the different

samples can be compared.

APPLICATIONS

• A standard for the study of gene expression at the level of

mRNA (messenger RNA transcripts)

• Detection of mRNA transcript size

• Study RNA degradation

• Study RNA splicing

• Study RNA half-life

• Often used to confirm and check transgenic / knockout mice

(animals)

Northern Application• Northern blots are particularly useful for determining the

conditions under which specific genes are being expressed, including which tissues in a complex organism express which of its genes at the mRNA level.

• For instance:

When trying to learn about the function of a certain protein, it is sometimes useful to purify mRNA from many different tissues or cell types and then prepare a Northern blot of those mRNAs, using a cDNA clone of the protein of interest as the probe. 

Only mRNA from the cell types that are synthesizing the protein will hybridize to the probe.

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Disadvantage of Northern plotting

1.The standard northern blot method is relatively less

sensitive than nuclease protection assays and RT-PCR

2. Detection with multiple probes is a problem

3. If RNA samples are even slightly degraded by RNases, the

quality of the data and quantitation of expression is quite

negatively affected.

Summary

Southern• DNA on membrane.• Digest DNA.• Convert dsDNA to

ssDNA.• Probe with DNA or RNA.

Northern• RNA on membrane.• No need to digest DNA.• Denature “folded” RNA

with formaldehyde.• Probe with DNA or RNA.

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Western blotting

• Western blotting (1981) is an Immunoblotting technique

which rely on the specificity of binding between a protein of

interest and a probe (antibody raised against that particular

protein) to allow detection of the protein of interest in a

mixture of many other similar molecules.

• The SDS PAGE technique is a prerequisite for Western

blotting .

Steps in western blotting

1. A protein sample is

subjected to

electrophoresis on an SDS-

polyacrylamide gel.

2. Electroblotting transfers

the separated proteins

from the gel to the surface

of a nitrocellulose

membrane.

Cont…

3. The blot is incubated with a generic protein

(such as milk proteins or BSA) which binds to

any remaining sticky places on the

nitrocellulose.

4. An antibody that is specific for the protein of

interest (the primary antibody - Ab1) is added

to the nitrocellulose sheet and reacts with the

antigen.  Only the band containing the protein

of interest binds the antibody, forming a layer

of antibody molecules .

Cont…

5. After washing for removal of non-

specifically bound Ab1, second

antibody (Ab2)is added, which

specifically recognizes  the Fc domain

of the primary antibody and binds it.

Ab2 is radioactively labeled, or is

covalently linked to a reporter

enzyme, which allows to visualize the

protein-Ab1-Ab2 complex.

An example

Application

1.The confirmatory HIV test

2.Western blot is also used as the definitive test

for Bovine spongiform encephalopathy (BSE(

3.Some forms of Lyme disease testing employ Western

blotting.

DOT-BLOTTING

• It is a modification of Southern and Northern blotting techniques described above.

• In this approach, the Nucleic acids (DNA or RNA) are directly spotted onto the filters, and not subjected to electrophoresis.

• The hybridization procedure is the same as in original blotting techniques.

• It is particularly useful in obtaining quantitative data for the evaluation of gene expression.

Autoradiography

• It is the process of localization and recording of a radiolabel within a solid specimen, with the production of an image in a photographic emulsion.

• These emulsions are composed of silver halide crystals suspended in gelatin.

• When a β-particle or a γ-ray from a radiolabel passes through the emulsions, silver ions are converted to metallic silver atoms.

• This results in the development of a visible image which can be easily detected.

Applications of autoradiography

• It is closely associated with blotting techniques for the detection of

- DNA

- RNA &

- Proteins.